1. Field of the Invention
This present invention concerns a medium for analytical and preparative electrophoresis.
2. Description of Related Art
Such aqueous solutions, which form a pH gradient upon high-voltage electrophoresis, are used on a large scale to analyze proteins and peptides. Such prior art solutions contain polyelectrolytes with amphoteric properties and are usually manufactured synthetically by means of condensation and/or polymerization of polyamines with unsaturated carboxylic acids. As a result of such synthesis, many different compounds with different molecular weights and a varying number of acid groups (carboxylic groups) and basic groups (amino and/or imino groups) are produced. Under the conditions prevalent during high-voltage electrophoresis, these polyelectrolytes arrange themselves, due to the different ratio between acidic and basic groups, in such a way that polyelectrolytes with particularly acidic properties concentrate in the proximity of the anode, and all other polyelectrolytes are arranged, in accordance with the “ranking” of acidic and basic properties, between the electrodes and are also concentrated in the process. By the selective accumulation of polyelectrolytes in accordance with the acidic and/or basic properties of the individual species, a pH gradient is produced throughout the entire aqueous solution.
By means of this pH gradient, amphoteric compounds such as proteins and peptides can be separated in accordance with the different acidic and/or basic properties. This analytical separation procedure is referred to in the literature as “isoelectric focusing”. In the situation where this procedure is carried out in a special gel (polyacrylamide), this procedure is referred to as PAGIEF (polyacrylamide isoelectric focusing). PAGIEF is considered the standard procedure.
There are a number of reported disadvantages when using the commercially available ampholytes. For example, the unsaturated acids that are used in the synthesis of commercially available ampholytes, e.g. substituted acrylic acids, are highly toxic. Their residual content is considered to represent a significant hazard. For that reason, the use of commercially available ampholytes for the cleaning of proteins for subsequent use in human applications is prohibited.
Additionally, during ampholyte synthesis, a large number of chemical species of unknown structure is formed whose relative concentration may vary significantly across batches. This has an adverse impact on the reproducibility of analytical results. In certain applications, “artificial bands” are reported as a result of undesired complex formation between the ampholyte and the analyte.
It has also been reported that partial interference with the analytical color reaction of proteins occurs which is caused by ampholytes with a higher molecular weight. Further, an unusually high percentage of ampholytes with a molecular weight>10 KD in products of certain manufacturers has been reported on several occasions. In the case of preparative cleaning of proteins, the subsequent separation of the ampholyte is difficult and sometimes incomplete; the suspected cause is complex formation. Finally, commercial ampholytes have been found to be unsuitable for use as separating media for bioparticles, considering that bioparticles aggregate in these media.